The present invention relates to compositions and methods for inhibiting activity of multimeric enzymes. In particular, it relates to inhibition of proteases by formation of dysfunctional protease multimers.
Protein structure is typically discussed in terms of four levels. The primary structure is the amino acid sequence; the secondary structure is any regular local structure of linear segment, such as an xcex1-helix; the tertiary structure is the overall topology of the folded polypeptide chain; and the quaternary structure is the aggregation of single polypeptides or subunits to form a functional molecule.
Many proteins exist as assemblies of two or more polypeptide chains, which may be identical or different. Complex interactions between the subunits are required to produce a functional protein. For example, multimeric enzymes can be rendered inactive, if the interaction of the monomers is disrupted.
Multimeric enzymes of particular interest to the present invention are multimeric proteases. Inhibition of protease activity may be useful in a number of contexts. For example, inhibition of retroviral proteases, which are critical to retroviral maturation and infectivity, can be used to inhibit retroviral infection.
Retroviruses are those viruses which have a single stranded RNA genome, a lipid envelope, and encode an RNA-dependent deoxyribonucleic acid (DNA) polymerase, known as reverse transcriptase. During their life cycle, the RNA genome is reverse transcribed into a DNA copy which is integrated into the genome of the host cell. A number of retroviruses cause disease states in humans. These include the lentiviruses, human immunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immune deficiency syndrome (AIDS), and the oncoviruses, human T-cell lymphotrophic viruses I and II which cause T cell leukemias.
Retroviruses, such as HIV-1, encode aspartic proteases that process polyprotein precursors into viral structural proteins and replicative enzymes that are essential for viral proliferation. Autoprocessing of the protease from the gag/pol polyprotein precursor results in the release of the protease and the generation of mature structural and enzymatic proteins derived from the gag and gag/pol polyproteins.
Studies of the crystal structure of HIV proteases (Navia, et al, Nature 337:615-620 (1989); Wlodawer, et al., Science 245:616-621 (1989)) have confirmed the homodimeric nature of these enzymes. Assembly of the two HIV protease monomers results in a dimer of approximately 22KD and generates an active site at the interface of the subunits.
These X-ray crystallographic studies have also defined regions of interaction between the monomers. Each monomer contributes half of the active site, which includes two catalytic aspartic acids as well as threonine/serine and glycine residues which are conserved among all aspartyl proteases for their structural role in maintaining active site geometry. The two N-termini and two C-termini of the individual monomers form xcex2-strands that interdigitate to create a four-stranded anti-parallel xcex2-sheet. These interactions appear to be a major stabilizing force in the enzyme and contribute over 50% of the inter-subunit contacts and hydrogen bonds. Dimer formation generates not only the catalytic center, but also the extended substrate binding pocket.
Since viral proteolytic activity is essential for the generation of infectious virus particles in HIV and related retroviruses, therapeutic intervention for HIV-1 and HIV-2 has targeted the HIV protease. Small molecules have been developed as inhibitors and are currently undergoing clinical trials as antiviral agents. In clinical trials however, resistance to these inhibitors is being observed. Thus, new approaches for inhibiting retroviral proteases, in particular HIV-1 protease, are an important therapeutic goal in the treatment of retroviral infections. The present invention addresses these and other needs.
The present invention provides methods of inhibiting multimeric enzymes, in particular proteases, both in vivo and in vitro. The methods comprise contacting a first protease monomer with a second, defective protease monomer, such that the defective monomer and the first monomer form a dysfunctional multimeric protease. If the target protease is associated with a disease state, the step of contacting may be carried out by administering the defective monomer to a patient. For example, a gene encoding the defective monomer may be administered in a retroviral vector suitable for gene therapy.
In some embodiments, the defective monomer is a defective human immunodeficiency virus (HIV) protease monomer. For example, a defective HIV protease monomer may be one in which an active site aspartic acid is replaced by a second amino acid residue, such as arginine, lysine, asparagine and the like. Alternatively the defective HIV protease monomer may be a monomer fragment, e.g., residues 6-99 of the HIV protease monomer.
Thus, the invention also provides methods of inhibiting HIV replication in a mammalian cell. The method comprises introducing into the cell a recombinant construct comprising a promoter sequence operably linked to a polynucleotide encoding a defective HIV protease monomer. The target cell may be a human hematopoietic stem cell.
The invention further provides recombinant constructs comprising a promoter sequence operably linked to a polynucleotide encoding a defective protease monomer, such as a defective HIV protease monomer. The promoter sequence may be, for example, from an HIV LTR.
The constructs may be incorporated in retroviral vectors capable of infecting mammalian cells. The invention thus provides mammalian cells (e.g., human T cells) comprising a recombinant expression cassette including promoter sequences operably linked to a polynucleotide encoding a defective protease monomer.
Definitions
As used herein a xe2x80x9cprotease monomerxe2x80x9d refers to a subunit of a multimeric protease that, when aggregated in the proper quaternary structure, results in the formation of an active protease.
As used herein the term xe2x80x9cHIV protease monomerxe2x80x9d refers to a monomer of approximately 99 amino acid residues, which results from autoprocessing of the HIV gag and gag/pol polyprotein precursors. The amino acid sequence and three-dimensional structure of an HIV protease monomer is described in Wlodawer et al. Science 245:616-621 (1989).
A xe2x80x9cdefective protease monomerxe2x80x9d is a mutated form of a protease monomer that is capable of forming a multimeric protease with one or more wild type monomers. The wild type monomers to which the defective monomer binds may be from the same or different proteases. Thus, the monomers of the invention can be used to inhibit the activity of a single protease or a family of related proteases. The multimeric protease so formed is dysfunctional, such that enzyme activity as determined in a standard assay for the particular protease is reduced by at least 50%, usually 75%, and preferably 90% or more, as compared to the wild-type multimeric enzyme.
A xe2x80x9cdefective HIV protease monomerxe2x80x9d of the invention refers to an HIV protease monomer which, when present in an HIV-infected cell, forms a heterodimeric or homodimeric dysfunctional HIV protease and which substantially inhibits HIV protease activity in an infected cell. HIV protease activity can be determined using an in vitro assay that measures the rate of specific hydrolysis of the decapeptide ATLNFPISPW, which corresponds to the sequence of the natural HIV-1 gag-pol polyprotein sequence from amino acid 151-160 and represents the junction between the protease and the reverse transcriptase. Cleavage occurs at the Fxe2x80x94P bond. A suitable protocol for carrying out this assay is described in Babe et al. Biochem. 30:106-111 (1991). HIV protease activity is substantially inhibited if activity as measured in this assay is reduced by at least 50%, preferably 75%, and most preferably 95% or more.
As used herein a xe2x80x9cpolynucleotide encoding a defective protease monomerxe2x80x9d is one which, when present in a cell, expresses a polypeptide which is or which comprises a defective protease monomer (e.g., HIV protease) of the invention. For example, the expressed polypeptide may be an isolated protease monomer or may be a polyprotein precursor (e.g., gag-pol) which is processed to produce the mature protease monoter.
In the expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be xe2x80x9csubstantially identicalxe2x80x9d to a sequence of the gene from which it was derived. For example, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the above term.
In addition, polynucleotides of the invention may encode polypeptides which are substantially identical to particular monomers disclosed here. Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term xe2x80x9ccomplementary toxe2x80x9d is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing subsets of the two sequences over a xe2x80x9ccomparison windowxe2x80x9d to identify and analyze local regions of sequence similarity. A xe2x80x9ccomparison windowxe2x80x9d, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms. Preferred programs include BLAST, OWL, or GenPept, using standard parameters.
xe2x80x9cPercentage of sequence identityxe2x80x9d is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term xe2x80x9csubstantial identityxe2x80x9d of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described above (e.g., BLAST) using standard parameters. one of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm of a hybrid, which is a function of both the length and the base composition of the probe, can be calculated using information provided in Sambrook, T. et al., (1989) Molecular Cloningxe2x80x94A Laboratory Manual, (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring. Typically, stringent conditions for a Southern blot protocol involve gashing at 65xc2x0 C. with 0.2xc3x97SSC.
Another indication that protein sequences are substantially identical is if one protein is specifically immunologically reactive with antibodies raised against the other protein. Under designated immunoassay conditions, specifically immunoreactive antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to defective HIV-1 protease monomers of the invention can be selected to obtain antibodies specifically immunoreactive with other defective monomers and not with other proteins. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
In the case of protease monomers of the invention, a modified protease monomer is substantially identical to a naturally occurring protease monomer if the modified protein is capable of binding the wild type monomer to form a heterodimer. Thus, modified proteins based on the monomers of the invention can be identified by assaying their ability to bind a wild type monomer to form a defective protease using assays described below.
The term xe2x80x9cretroviral vectorxe2x80x9d as used herein means a vector that is derived from a retrovirus. Retroviral vectors may be constructed to have the capability to insert a gene or DNA fragment into the host chromosomal genome by a recombinational event, such that the DNA fragment may be stably expressed in the host cell. The basic design and use of retroviral vectors is described, for instance, in Singer, M. and Berg, P. Genes and Genomes, Mill Valley, Calif. (1991) pp. 310-314. A retrovirus is present in the RNA form in its viral capsid and forms a double-stranded DNA intermediate when it replicates in the host cell. Similarly, retroviral vectors are present in both RNA and double-stranded DNA forms, both of which forms are included in the term xe2x80x9cretroviral vectorxe2x80x9d.
The term xe2x80x9cgene therapyxe2x80x9d as used herein refers to a method of treating disease in an animal by introducing a nucleic acid (either RNA or DNA) into the cells of the animal. The nucleic acid introduced may be from the same species as the animal being treated or it may originate from another species. The nucleic acid may also be synthetically produced. The term includes introduction of nucleic acids into specific cells of the animal where the nucleic acids are subsequently expressed in the animal""s cells and where the result of this expression is treatment of a disease.
As used herein, the term xe2x80x9coperably linkedxe2x80x9d refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is xe2x80x9coperably linkedxe2x80x9d when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
A xe2x80x9crecombinant expression cassettexe2x80x9d is a polynucleotide sequence containing a coding sequence which is capable of affecting expression of the coding sequence in hosts compatible with the sequence. Such cassettes include the coding sequence and regulatory sequences such as promoters, transcription termination signals as well as other sequences (e.g. enhancers) necessary or helpful in affecting expression.